Future Goals

• Repeat the Construction of Dry Forest Library
• Optimization of the electroelution step (critical step)
• Construct a library from Microbial Mats and El Yunque dry forests
• Plasmid Extraction of the clones that were obtained

DNA Extraction & Library Construction

We decide to do again the library from the beginning. Some changes have been performed to the protocol for the DNA extraction using the Freeze and Thaw method. The soil sample was divide in four bottles and the quantities of the different solutions that were going to be used were readjust. 

Again, we used GITC (guanidine isothiocyanate) instead of Guanidine HCl and because of the lack of Tris buffered phenol chloroform we only used chloroform. After the electroelution we tried to determine the DNA concentration. For each tube we made 3 readings but the values were not consistent and some negative values were obtained.  We decide to use randomly one of the tubes from the electroelution (not the rinse). The DNA was concentrate into a final volume of 100uL but it concentration was not read because of the problems with the other lectures. For the end repair step, 40uL of DNA was taken directly from this tube instead of 40uL of the supposed dilute DNA (0.5ug/uL) from this tube. The other steps of the CopyControl^TM Fosmid Library Production Kit protocol were done as directed. Next they, after the inoculation of the possible clones, we can observed some colonies in the plates!!!!

Library Construction

For the library construction we used the DNA extracted from the electroelution that was preserved at -20^oC. DNA concentration was measured again with the spectrophotometer to verify if we had DNA. The obtained DNA concentrations were different from the last time we measure it. The concentrations obtained were 15, 190, 55 and 3.3ng/uL for the tubes of 1, 1R, 2, and 2R respectively. It was strange, the fact that in R2 (R2 stands for the rinse of the DNA obtained after the electroelution of noodle 2) the concentration of DNA was greater than tube 1 and very similar to tube 2.  For the Library construction we decide to use 2R. Before the library construction we concentrate the DNA obtaining a final DNA concentration of 1350ng/uL. After this, we proceed with the library construction following the instructions of the CopyControl^TM Fosmid Library Production Kit. For the growing of the possible infected cells or clones we use LB agar with 12.5ug/mL of chloramphenicol and we did it too with a lower chloramphenicol concentration of 6.5ug/mL (were made by mistake). We growth the EPI300 (not resistant to cloramphenicol) in 6.5ug/mL of chloramphenicol to verify if it was capable of growth but it was not.  The plates in which the possible clones were infected with non dilute phages and the dilution of 1:10 show some colonies growth, but it was only in the plates of the lower concentration of chloramphenicol (6.5ug/mL). To verify the chloramphenicol resistance we inoculate the possible clones in 12.5ug/mL of chloramphenicol and were incubate overnight. Not all the transferred colonies growth. The growth ones were preserved and a plasmid extraction is going to be performed to confirm and see if it has the vector and the DNA insert.

February 5, 2011

RESULTS

February 4, 2011

ELECTROPHORESIS GEL:

We realize an electrophoreis gel to confirm the DNA presence

February 1, 2011

NEW!! FREEZE AND THAW:

We decide to work with new samples.

Soil Description:
B #2= sandy, reddish color, abundance of plant material, 45˚C

E #5=slimy soil, brown color, abundance of plant material, 39˚C

C #3=clayey soil, dark brown color, abundance of plant material, 51˚C

 

At the end of the extraction we decide to realize a master DNA with all of them.  It will help us to create a Metagenomic Library

January 31, 2011

We come back from Christmas Holidays:

-We revise again all the reactive and we determine which one was missing
-We concentrate the DNA that we have obtained from the last experiment

DNA concentrations determined:

         6 Rinse= 40ug/ul

         5 (2)= 60mg/ul

December 22, 2010

We can't realize the End- repair protocol because we don't have the enough End- Repair reactive

December 21, 2010.

ELECTROELUTION CONCENTRATIONS:

5=165ng/ul

5Rinse=40ng/u

6=115ng/ul

6Rinse=125ng/ul

December 1, 2010.

ELECTROELUTION

-We had substitute the Agar ACE with GELase
-4˚C Overnight

DNA concentrations:

S1=45ng/ul

S2=145ng/ml

November 3, 2010.

Oct. 26, 2010. SIZING GEL Modifications: Ladders: 40kb and Lambda Hind III

5= Bottle 5

6= Bottle 6

 L1= Hind III

L2= 40kb

October 27, 2010.

SIZING GEL Modifications: Ladders: 40kb and Lambda Hind III

1= Bottle 1

2= Bottle 2

 L1= Hind III

L2= 40kb

3= Bottle 3

4= Bottle 4

September 29, 2010.

Chaotropic buffer substitution:

-Guanidine isothiocyanate (GITC) with Guanidine HCl

                G-HCl 5M in 20ml

October 4, 2010.

Bottles	Soil (g)	Z Buffer (ml)
1	15.028g	22.5ml
2	15.12g	22.5ml
3	15.13g	22.5ml
4	15.05g	22.5ml
5	15.07g	22.5ml
6	15.05g	22.5ml

**Freeze and Thaw**

October 9, 2010.

DNA Purification

September 27, 2010.

First of all we optimize the protocol based on the amount of sample and other materials that we had for work.

-We prepared stocks of:

CTAB  100ml and EDTA  500ml

-Computations for use 50ml bottles for the Freeze and Thaw:

	Original	Modified	Total (min)
bottle	250ml	50ml
Soil	50g	15g	90g
Z buffer	75ml	22.5ml	135ml
SDS	9ml	2.7ml	16.2ml
GITC	4.5ml	1.35ml	8.1

·         The intention is to extract DNA from soil using three 50ml bottles with 15g of soil in each one.

We will have:

6 bottles=90g of soil

September 28, 2010.

-We prepared stocks of:

NaCl (5M)

Sodium Phosphate solution

Tris HCl (1M)

Z Buffer

(100ml sterile Tris, 50ml sterile Sodium Phosphate Solution, 200ml sterile EDTA, 300ml sterile NaCl, 100ml CTAB 10%, 250ml Water dH₂O)

SDS 20 %

                We substitute the Lauryl with Sodium dodecyl sulfate